JPS61205482A - Tissue culture of thalictrum thunbergii - Google Patents

Abstract

PURPOSE:To obtain a large amount of useful berberine, in high efficiency, by the tissue culture of Thalictrum thunbergii (a kind of larch tree) in a medium containing a specific plant hormone by two-stage cultivation process. CONSTITUTION:A selected culture cell is cultured in a medium containing a plant hormone composed essentially and solely of plant hormones selected from phenoxyacetic acid-type auxin (the first cultivation step). The cultured cell is transferred to the medium containing a plant hormone essentially free from the plant hormones selected from phenoxyacetic acid-type auxin, and cultured (the second cultivation step). The cell is grown stably, and the cultured cell of thalictrum thunbergii having high berberine-productivity can be produced.

Description

[Detailed Description of the Invention] [Technical Field of the Invention] The present invention relates to
1nusL, var, Sokou Eayuμ徂Miq, -T,
The present invention relates to a tissue culture method by Dr. For more details, see 2 using a medium containing specific plant hormones.
This invention relates to a method for efficiently producing a large amount of berberine, a useful ingredient, by tissue culturing Japanese larch using stage culture.

[Conventional technology]

Japanese larch, a plant in the Ranunculaceae family, contains berberine, and this alkaloid is used as an intestinal medicine, for example.
It is used as a treatment for bacterial intestinal diseases, as a dye, and is in high demand.

However, the method of directly collecting berberine from naturally grown Japanese larch is difficult because the berberine content of the Japanese larch plant is extremely low, its growth is affected by the natural environment and weather, and it takes time and effort to collect the plant. It cannot be said to be an advantageous method because it takes a lot of time.

Therefore, as an alternative to these methods, methods for propagating Japanese larch using tissue culture methods have been proposed, such as phytochemistry (Phyto-'chemis).
Try) Vol. 21, pp. 1419-1421 (1982) describes Murashig containing 2,4-dichlorophenoxyacetic acid and kinetin as plant hormones.
A method for tissue culturing Japanese larch using e-3koog's solid medium has been proposed. According to this method, the amount of berberine produced is 0.0019% per dry weight in natural Japanese larch, whereas an MS medium containing 1.0 ppm and 5.0 ppa+ of 2,4-dichlorophenoxyacetic acid was used. It is stated that callus obtained by tissue culture contains 0.25% and 0.67% berberine, respectively. However, even with this method, the amount of berberine produced is still insufficient, and in order to industrially produce berberine using tissue culture, a medium that can obtain cultured cells with a higher berberine-producing ability than conventionally known ones is required. It is necessary to develop

Under such circumstances, the applicant of the present invention previously filed an application in Japanese Patent Application No. 82951/1983 for an excellent method for obtaining cultured cells whose berberine-producing ability is significantly higher than that of conventionally known cells.

By the way, it is generally known that in plant tissue culture, the properties of growing cultured cells are influenced by the type of medium used, such as the component composition of the medium and the content of the components. The method previously proposed by the present inventors still leaves room for further study.

[Purpose of the invention]

Recognizing this point, the present inventors further investigated a tissue culture method for obtaining cultured Japanese larch cells that are more stable, have better growth, and have higher productivity of berberine than ever before.

[Summary of the invention]

As a result, the inventors discovered that the above object could be achieved by employing the following method, and completed the present invention.

That is, according to the method of the present invention, when selectively cultured cells derived from Japanese larch tissue are tissue cultured using a medium, the selectively cultured cells are treated with phenoxyacetic acid as a plant hormone in the first step of the culture. The cultured cells are cultured in a medium containing only a plant hormone selected from auxin, and then, in the second stage of the culture, the cultured cells are cultured by the method of the first stage.The plant hormone is substantially selected from phenoxyacetic acid auxin. Provided is a tissue culture method for Japanese larch, which comprises culturing in a medium that does not contain plant hormones.

In the method of the present invention, tissue culture of Japanese larch is carried out in two stages: a first stage and a second stage.

The details of this method will be explained below.

[First stage tissue culture]

In the method of the present invention, a Japanese larch tissue is cultured by a known subculture method described below to select cultured cells having the ability to grow berberine, and the first step of the tissue culture of the present invention is performed using these selected cells. be exposed. That is, in the first stage of tissue culture of the present invention, the previously selected cultured cells are tissue cultured using a medium containing only plant hormones selected from phenoxyacetic acid auxins as plant hormones. In this case, two or more kinds of the plant hormones may be used in combination, if necessary.

In the tissue culture of the first stage of the present invention, the plant hormones selected from phenoxyacetic acid auxins used as plant hormones include 2.4-dichlorophenoxyacetic acid, 3.4-dichlorophenoxyacetic acid, and 2-methyl- 4-chlorophenoxyacetic acid, 2.4.5-)dichlorophenoxyacetic acid, 2-(4-chlorophenoxy)
Propionic acid, 2-(2-methylphenol).

xy)propionic acid, 2-(2,4-dichlorophenoxy)propionic acid, 2-(3,4-dichlorophenoxy)propionic acid, 2-(2-methyl-4-chlorophenoxy)propionic acid, 2-(2 , 4,5-trichlorophenoxy)propionic acid, γ-(4-chlorophenoxy)butyric acid, γ-(2,4-dichlorophenoxy)butyric acid,
r-(3,4-dichlorophenoxy)butyric acid, γ-(2
-methyl-4-chlorophenoxy)butyric acid, β-(2,4
-dichlorophenoxy)acrylic acid, α-(2-methyl-4-chlorophenoxy)phenylacetic acid M, S-ethyl (
4-chloro-2-methylphenoxy)thioacetate,
2,4-dichlorophenoxyacetohydrazide, 2-methyl-4-chlorophenoxyacetohydrazide, N-methoxy-(2-methyl-4-chlorophenoxy)acetamide, N-phenyl-(2-methyl-4-chlorophenoxy) Acetamide, N-(2-chlorophenyl)(
2-Methyl-4-chlorophenoxy)acetamide, N
-(3-trifluoromethylphenyl)(2-methyl-
4-chlorophenoxy)acetamide, 2-methyl-4
-chlorophenoxyethanol, 2- (2,4,5-
) dichlorophenoxy)ethyl-2,2-dichloropropionate, 2,4-dichlorophenoxyethylbenzoate, methyl-α-naphthoxyacetate, 2-(1
-naphthoxy)N,N-diethylpropionamide, α
-(β-naphthoxy)propionanilide and the like can be exemplified.

Among the above-mentioned plant hormones selected from the phenoxyacetic acid auxins used as plant hormones in the first stage tissue culture of the present invention, 2,4-dichlorophenoxyacetic acid, r-(2,4-dichlorophenoxy)butyric acid, and ,
Preference is given to using γ-(2-methyl-4-chlorophenoxy)butyric acid and 2-methyl-4-chlorophenoxyacetic acid.

The medium used in the first step of the culture method of the present invention contains at least one type of plant hormone selected from the group of phenoxyacetic acid auxins described above as an inorganic component, a carbon source, and a plant hormone (for convenience, these plant hormones are hereinafter referred to as phytohormones). This medium contains plant hormones (referred to as A)) as an essential component, to which vitamins are added, and amino acids are further added as necessary. Inorganic components of the medium include inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate. Examples include compounds such as manganese sulfate, copper sulfate, sodium molybdate, molybdenum oxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Substantially only the above-mentioned plant hormone (A) is used as the plant hormone in the medium. What I mean here is essentially
A plant hormone added to a culture medium in which the amount of other commonly known plant hormones other than the plant hormone (A) is zero or, if present, the amount is usually 10 M or less. This means that the amount is much smaller than the amount of , and does not substantially affect the culture.

The vitamins in the medium include biogin, thiamine (vitamin Bl), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin D2).

Examples of amino acids in the medium include glycine, alanine, glutamic acid, cysteine, tyrosine, and lysine.

The medium of the present invention usually contains 0.17% of the inorganic component.
7M to about 100a+M, about 1 g/l of said carbon source
to about 100 g/l, usually 0.01 MM to 1000 μM, preferably 1 μH of said plant hormone (A).
It contains about 0 to 100 μM of the vitamins.
．． 1a+g/j' to about 150 mg/1 and the above amino acids in O to about 1000 H/j! It is desirable to use it together.

Specifically, the medium used for tissue culture of Japanese larch of the present invention may be a culture medium conventionally used for tissue culture of plants, such as Murashige & Skoog ('62).
), Linsmeyer-Skoog (RM-196
5) Add the carbon source and plant hormone (A) described above to (Lins+waier & Skoog) medium, White medium ('63), Gaa+borg B-5 medium, Mitsui M-9 medium, etc. Examples include a culture medium prepared by adding the above vitamins and amino acids as necessary, but in the present invention, in particular, a Linsmeyer-Skoog or Murashige-Skoog medium is used. A medium is preferred. The composition of the conventionally known culture medium described above is described, for example, in "New Plant Tissue Culture JP 388-P391," by Yukinai, Nakajima, and Furuya, Asakura Shoten, 1979.

The medium that can be used in the present invention is a liquid medium or a solid medium containing usually 0.5 to 1% agar, but it is preferable to use a liquid medium in the present invention.

In the first stage of tissue culture of the present invention, light is not necessarily required, and culturing in the dark is desirable for the growth of cultured cells.

The culture temperature in the culture medium of the present invention is usually 20 to 30°C, but 23 to 28°C is particularly preferable, and even if the temperature is lower or higher than this, the growth rate of the cultured cells will not change. descend.

In the first stage of the tissue culture of Japanese larch of the present invention, the cultured cells proliferate and grow, and stable cultured cells are obtained. In particular, by employing the first stage culture method of the present invention, stable cultured cells with thick cells (excellent growth) can be obtained, which means that the second stage of the present invention using the cultured cells described below It is important in tissue culture because it has a great effect on significantly increasing the production amount of berberine, a metabolite of cultured cells.

[Second stage tissue culture]

In the method of the present invention, after the tissue of Japanese larch is cultured by the above-described first stage tissue culture method, the cultured cells are further cultured by the second stage tissue culture method to produce berberine. In the tissue culture of the second stage of the present invention, the plant hormone is substantially the above-mentioned plant hormone (A
) is cultured using a liquid medium that does not contain

The fresh medium used in the second stage of the culture method of the present invention has inorganic components and carbon sources as essential components, to which are added plant hormones and vitamins, and further added with amino acids as necessary. It is a medium.

Examples of the inorganic components and carbon sources of the medium include those used in the first stage tissue culture of the present invention described above.

Examples of the plant hormones in the medium include auxins such as naphthalene acetic acid (NAA), indole acetic acid (IAA'I), and indole butyric acid (IBA), and cytokinins such as benzyladenine (BA), kinetin, and zeatin. A group of these plant hormones used in the second stage tissue culture of the present invention is hereinafter referred to as plant hormones (B).In the present invention, a fresh medium used in the second stage culture is prepared. In this case, the above-mentioned plant hormone (A) is not used as the plant hormone. In the present invention, when the above-mentioned plant hormone (B) is used, it is more preferable to use the above-mentioned auxins and the above-mentioned cytokinins together. In this case, it is particularly preferable to use NNA and BA because they increase the amount of berberine produced.

Examples of the vitamins and amino acids in the medium include those used in the first step of tissue culture of the present invention described above.

The medium used in the second stage of tissue culture of the present invention usually contains about 0.1 μA to about 100 mM of the inorganic component and about 1 g/f to about 100 μM of the carbon source.
g/l, about 0.01 p of said plant hormone (B)
H to about 1000 μA, especially NAA, usually about 1 μH to about 1000 μH, preferably about 50
μH to about 100 μM, BA usually about 0.1 μM
H to about 100 μH2 preferably about 1 μH to about 1
The amount of the vitamins is about 0.1 mg/l to about 150 mg/It, and the amino acids are about 0 to about 1000 a+g/12.

Specifically, the medium used in the second stage of tissue culture of the present invention is a conventionally known medium used in the first stage of tissue culture of the present invention, and the above-described carbon sources and plant hormones,
Furthermore, examples include a medium prepared by adding the above-mentioned vitamins and amino acids as necessary, but in the present invention, a medium prepared using a Linsmeyer-Skoog or Murashige-Skoog medium is particularly preferred.

The medium that can be used in the present invention is a liquid medium or a solid medium containing usually 0.5 to 1% agar, but in the present invention, it is preferable to use a liquid medium as in the first stage.

In the second stage tissue culture of the present invention, the cultured cells obtained by the first stage tissue culture of the present invention are transferred to the medium used in the second stage tissue culture described above. In this case, the fresh medium does not contain the plant hormone (A) as described above, but when transferring the cultured cells from the first stage medium to the second stage medium, the cultured cells are washed with water, for example. However, a small amount of the plant hormone (A) may adhere to or be incorporated into the cultured cells and contaminate the medium used in the second stage of culture.

However, this contamination usually results in plant hormones (A
) is extremely low compared to the lower limit of the concentration of plant hormones in the fresh medium used in the second stage culture, and is at a level where the influence on the culture can be ignored. The medium used in tissue culture is expressed as a medium substantially free of plant hormone (A).

In the second stage of tissue culture of the present invention, light is not necessarily required, and culture in the dark is preferable for growing cultured cells.

The culture temperature in the culture medium of the present invention is usually 20 to 30°C, but 23 to 28°C is particularly preferable, and even if the temperature is lower or higher than this, the growth rate of the cultured cells will not change. descend.

In the present invention, berberine is produced as a metabolite in the second stage of culturing the Japanese larch tissue from the cultured cells that have grown sufficiently in the first stage of culturing. By employing the first and second stage tissue culture methods of the present invention, berberine can be obtained more stably and efficiently in larger amounts than in the past.

[An example of tissue culture of the present invention]

As a typical example of the tissue culture method of the present invention, the method described below can be exemplified. That is, tissue pieces collected from roots, leaves, stems, seeds, and flower buds of Japanese larch were sterilized, placed on a Linsmeyer-Skoog solid medium solidified with agar, and incubated at 20 to 30°C for 20 to 30 minutes. Leave it for about a day to allow some of the tissue pieces to form a callus. By subculturing the callus obtained in this way using, for example, a specific liquid medium developed by the present inventors (presented in Japanese Patent Application No. 59-82952), berberin, a metabolic product of Japanese larch cells, can be extracted. It is possible to obtain stable selected cultured cells with a fast growth rate that have the property of releasing ions to the outside of the cells. In the method of the present invention, if this stabilized berberine-releasing selected cultured cell is used in the tissue culture method of the present invention, it is convenient to separate and recover berberine from the culture mixture. The method is not limited to the method of using releasable selectively cultured cells, but by subculturing the callus using a commonly known medium, stable culture with high berberine productivity and fast growth rate can be achieved. Cells can be selected and the selected cultured cells can be used in the tissue culture method of the present invention.

In the present invention, the berberine productivity obtained by the above-mentioned subculture is high (fast growth rate, stable selected cultured cells), and this is used in the first and second stage tissue culture of the present invention. By culturing according to the method described above, a culture mixture containing cultured cells, berberine, and a medium is obtained.

[Separation of berberine]

In the tissue culture method of the present invention, especially when selectively cultured cells are used in which berberine is released from inside the cells into the medium, approximately 80% or more of the total amount of berberine produced is usually released outside the cells. In this case, since the nitrate or chloride of berberine is a salt that is H-soluble in cold water, in one embodiment of the present invention, ammonium nitrate, ammonium chloride, etc. By using a liquid medium containing nitrate ions and chloride ions, the released berberine can be precipitated as yellow crystals of berberine nitrate and berberine chloride. In this case, the released berberine may exist in a dissolved form in the liquid medium in addition to the yellow precipitated crystals. Therefore, as a method for separating berberine, for example, appropriate amounts of nitrate ions and chloride ions are added to the culture mixture after the cultivation is completed, and the dissolved berberine is precipitated by cooling to an appropriate temperature, and then the yellow precipitated crystals are picked up from the liquid medium. It is also possible to adopt a separation method different from conventional methods such as extraction.

In one embodiment of the present invention, the culture medium used in the second stage of the tissue culture of the present invention has a concentration of inorganic components such as phosphates, such as potassium phosphate, sodium phosphate, etc.
70mg/It or less, preferably 100mg/1
When tissue culture is performed using berberine-releasing cultured cells using the following liquid medium, a favorable phenomenon occurs in which the growth of the cultured cells is suppressed and the amount of berberine released to the outside of the cells increases. The inventors have discovered that.

In the method of the present invention, when a liquid medium is used, as a method for separating berberine obtained by tissue culture, for example, the method described below can be adopted as necessary.

(1) After the completion of the culture, the culture mixture containing the cultured cells and the metabolite berberine is heated to dissolve berberine in the liquid medium in the culture mixture, and the mixture is separated into a solution and a solid by a method such as filtration, Next, the solution is usually cooled to below 10°C to precipitate berberine as yellow crystals. In this case, if necessary, nitrate ions or chloride ions may be added to the solution to precipitate berberine as yellow crystals of nitrate or chloride. The yellow precipitate is then isolated by a method such as filtration.

(2) Remove the solid mixture of cultured cells and yellow crystal precipitate from the liquid medium by, for example, centrifuging the culture mixture or filtering the culture mixture using a nylon or metal filter with an appropriate pore size. After separation, the solid mixture is treated with a solvent such as chloroform, methanol, or ethanol to extract berberine into the solvent and crystallized to isolate berberine. On the other hand, the previously separated liquid dissolves berberine, which is, for example, A+
After adsorption onto an ion exchange resin such as wberlite XAD-2, berberine is isolated by elution with methanol and crystallization. Other methods for isolating berberine dissolved in the separated liquid include extracting and separating the separated liquid by treating it with a solvent such as chloroform, or the method described in (1) above. ), it is also possible to adopt a method of adding nitrate ions and chloride ions to the liquid to precipitate and separate berberine.

(3) If the produced berberine remains in the cells, separate the cultured cells from the culture mixture by a method such as filtration similar to (2), crush the cultured cells, and then extract and separate the berberine. It is also possible to adopt the method of

〔Effect of the invention〕

If the tissue culture method for Japanese larch of the present invention is adopted, the culture will be performed using stable cultured cells that have grown sufficiently through the first stage of tissue culture of the present invention, so the productivity of berberine will be improved compared to the conventional method. It is industrially useful because it makes it possible to reliably mass-produce berberine.

〔Example〕

Hereinafter, the method of the present invention will be explained in more detail with reference to Examples.

Examples 1 to 4 Leaves, petioles, and flower buds of Japanese larch were sterilized in a conventional manner,
NAA 100μM, BA 5μH and sucrose 30g/j! Calli were induced from these by a conventional method using a Linsmeyer-Skoog solid medium containing a concentration of . Since callus growth and berberine production ability differ depending on the tissue of the Japanese larch used, the callus was allowed to grow and divide in the dark at 25°C using the above-mentioned Linsmeyer-Skoog solid medium, resulting in deep yellow growth. By subculturing the calli for about one year while selecting calli with good berberine, stable calli with a deep yellow color and high production of berberine were obtained.

Next, the selected callus was treated with 100 μM of NAA and 5 μM of BA.
μh and sucrose at a concentration of 30 g/l into a Sunsmeyer-Skoog liquid medium at a temperature of 25
℃, subcultured under shaking conditions of 100 strokes/min,
Cultured cells with deep yellow color and good growth were selected using a Komagome pipette with a thick tip, transferred to fresh liquid medium, and cultured again. In this case, the number of culture days after the selected cells are transferred to a fresh liquid medium is 2 to 3 weeks, and by repeating this selection and subplanting about 5 times over 2 to 3 months, a large amount of berberine is produced. Moreover, it was possible to obtain berberine-releasing cultured cells that release more than 80% of the produced berberine to the outside of the cells.

Next, using a 100 ml Erlenmeyer flask, the berberine-releasing cultured cells were cultured in Examples 1 to 1 in Table 1.
Culture at 25°C and 100 strokes/min in a Linsmeyer-Skoog liquid medium containing 30 g/n of various phenoxyacetic acid auxins and sucrose as plant hormones shown in the first stage culture column of 4. When the cells were cultured for 14 days with shaking, the growth rates shown in the first stage culture column of Examples 1 to 4 in Table 1 were obtained.

Here, the growth rate is a value calculated by (dry weight of cells obtained at the end of culture/dry weight of seed cells).

Next, the cells obtained in the first stage of culture were each incubated at a concentration of 1.
00 μH NA^ and 1 μH BA and sucrose 30 g/j! Cultured for 14 days in a Linsmeyer-Skoog liquid medium containing the following conditions at a culture temperature of 25°C and shaking at 100 strokes/min. The indicated growth rates and berberine contents were obtained. Table 1 also shows berberine productivity, which is expressed as the product of the first stage growth rate, second stage growth rate, and berberine content.

Comparative Examples 1 to 5 Cells were cultured under the same culture conditions as in Examples 1 to 4, except for the plant hormones used in the first and second stage cultures. As a result, the growth ratio and berberine content shown in Table 1 were obtained, and when the berberine productivity was calculated from them, all of them were lower than the berberine productivity obtained in Examples 1 to 4.

Claims (1)

[Claims] (1) When selectively cultured cells derived from Japanese larch tissue are cultured using a medium, the first step of the culture is to use only plant hormones selected from phenoxyacetic acid auxins as plant hormones. and then, in the second step of the culture, the cultured cells cultured by the method of the first step are cultured in a medium containing a plant hormone containing a plant hormone substantially selected from phenoxyacetic acid auxins. A method for culturing tissues of Japanese larch, which is characterized by culturing in a medium containing no.