JPS60227673A - Method for selecting cultivated cell of thalictrum thunbergii dc. - Google Patents

Abstract

PURPOSE:To obtain cultivated cells capable of releasing berberine useful as a medicine for intestinal disorders, etc., to the outside of the cells, by subculturing cultivated cells of Thalictrum thunbergii DC. in a culture medium containing benzyladenine while selecting deep yellow cultivated cell among the cultivated cells. CONSTITUTION:A solid or liquid culture medium containing benzyladenine as an essential component and further an inorganic component and carbon source and if necessary and amino acid, etc. added thereto is prepared. Cultivated cells of Thalictrum thunbergii DC. are subcultured in the resultant culture medium while selecting deep yellow cultivated cells among the cultivated cells to give the aimed berberine-releasing cultivated cells capable of releasing berberine. The resultant cultivated cells are multiplied and cultivated in a liquid culture medium, and the produced berberine is isolated from the cultivation mixture by a method of filtration, extraction with a solvent, column chromatography, etc. to afford advantageously the aimed berberine.

Description

[Detailed Description of the Invention] [Technical Field of the Invention] The present invention relates to
u m 11 Lee L, v a r, h1 animal] e
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This invention relates to a method for selecting cultured cells. More specifically, by subculturing Japanese larch tissue using a liquid medium with a specific composition, cultured cells with the property of releasing the useful components of berberine to the outside of the cells were obtained, and the cultured cells were tissue cultured to produce berberine. Regarding how to get .

[Industrial application field]

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

[Conventional technology]

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 so far, such as phytochemistry (phytochemistry).
ry) Vol. 21, pp. 1419-1421 (1982) describes Mu containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as plant hormones.
A method of tissue culture of Japanese larch using a solid medium of Rashige-3 Koog has been proposed.

However, with these conventionally known methods, the amount of berberine produced by tissue culture is small, and since berherine exists within cells, it is difficult to extract berberine from the nutrient mixture after culturing. For separation, it is necessary to separate the cultured cells from the medium by a method such as decantation or filtration, and then perform a process such as crushing the cultured cells to remove berberine from the cells. Operation is complicated.

[Purpose of the invention]

Recognizing this current situation, the present inventors have intensively investigated a method that can increase the amount of berberine produced by tissue culture and can separate berberine using a method that is simpler than conventional methods.

[Structure of the Invention] As a result, the following method was discovered and the present invention was completed.

That is, according to the present invention, berberine is released to the outside of the cells by subculturing the cultured Japanese larch cells while selecting the dark yellowish cultured cells using a liquid medium containing benzyladenine. A method for obtaining cultured Japanese larch cells is provided.

By adopting the method of the present invention, berberine-releasing cultured Japanese larch cells that selectively produce ruberin and have the property of releasing the produced berberine to the outside of the cells can be obtained. In this case, this property can be used to produce berberine using a simpler method than conventional methods. The existence of cultured Japanese larch cells that have the ability to release berberine was newly discovered by the present inventors, and the cultured cells have the characteristic that they are not destroyed even if they release berberine. .

The tissue culture medium used for subculturing the cultured Japanese larch cells of the present invention contains an inorganic component and a carbon source as essential components, and also contains benzyladenine (hereinafter sometimes abbreviated as BA) as an essential component. plant hormones,
This is a medium to which vitamins are added and, if necessary, amino acids are added.

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, mono-lithium dihydrogen phosphate,
Examples of compounds include magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride. can.

Examples of carbon sources for the +8 pond include carbohydrates such as silosaccharide and their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol.

In the present invention, it is an essential condition that the medium contains BA as a plant hormone, and other plant hormones such as naphthalene acetic acid (NAA), indole acetic acid (I), etc.
AA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D) indolebutyric acid (IB
Although appropriate amounts of auxins such as A) and cytokinins such as kinetin and zeatin may be added as necessary, it is particularly preferred to use BA and NAA in combination in the method of the present invention.

The vitamins in the medium include biotin, thiamine (vitamin B, pyridoxine (vitamin BI), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotine Examples include acid amides and riboflavin (vitamin B, >).

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

The medium used for the above-mentioned subculturing of the present invention usually contains about 0.1 μM to about 100 mM of the inorganic component, about 1 g/β to about 100 g/R of the carbon source, excluding BA among the plant hormones. 0 to about 2 plant hormones
00μM1 The above vitamins are about 0.1 mg/II to about 150 mg//! and 0 to about 1000 mg/ρ of the above amino acids.

The medium of the present invention is a liquid medium containing the above-mentioned components or a solid medium obtained by adding, for example, about 1% agar to the liquid medium, and among these, it is particularly preferable to use a liquid medium.

Specifically, the medium used for subculturing the red larch of the present invention may be a conventionally known medium used for tissue culture of plants, such as Murashige & Skog ('62). -oo
g) medium, Linsmeyer-Skoog (RM196
5) CLinsmaier &Skoog's medium, White's medium, Gam-borg's B-5 medium, Mitsui's M
An example of a TM-manufactured medium is obtained by adding a plant hormone containing BA as an essential component and a carbon source to a -9 medium, and further adding the above-mentioned vitamins and amino acids as necessary. Among these, a liquid medium prepared from a Linsmeyer-Skoog medium is particularly preferred. Regarding the composition of the above-mentioned conventionally known culture medium, for example, see "New Plant Tissue Culture J P386-P3" by Otsuchi, Nakajima, and Furuya.
91 Asakura Shoten, 1979.

In the solid or liquid medium of the present invention, the amount of BA added is usually 0.1 μM to 100 μM, preferably 0.5 μM.
Excellent cultured cells can be obtained when the concentration is in the range of μM to 10 μM.

As an example of the method for subculturing the Japanese larch tissue of the present invention, the following method 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 30 days to form a callus in a part of the tissue piece. By subculturing the callus thus obtained using the solid or liquid medium of the present invention, 80% of the produced berberine, which is a metabolic product of the Japanese larch cells, is
It is possible to obtain stable cultured Japanese larch cells with a high growth rate and a property of releasing more than % of the cellulose to the outside of the cells. In this case, the following method can be specifically exemplified as the subculture method of the present invention. From the callus obtained by culturing the above-mentioned callus-formed tissue in the solid medium of the present invention, dark yellow cultured cells that produce a large amount of berberine are collected using, for example, tweezers.
Use a spatula or the like or make the tip thicker (2~
5 mm) using a Komagome pipette, etc., transfer the cultured cells to the fresh liquid medium of the present invention and further culture, and select deep yellow cultured cells from the obtained cultured cells in the same manner as before. The method of subculture is repeated by repeating the culture in fresh liquid medium.

By this method, cultured Japanese larch cells having the property of releasing berberine to the outside of the cells, which is the object of the present invention, can be obtained.

In subculturing the Japanese larch 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, particularly 23 to 28°C.
C. is preferred; if the temperature is lower or higher than this, the growth rate of the cultured cells will decrease.

For example, the following method can be exemplified as a specific method for producing berberine according to the present invention. The berberine-releasing cultured cells of the present invention were cultured in a liquid medium in the dark at a temperature of 2.
A culture mixture is obtained by growing at 0 to 30°C. As a method for separating berberine from the culture mixture, for example, the method described below can be adopted as necessary.

(1) After the cultivation is completed, 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 a yellow solid of nitrate or chloride. The yellow precipitate is then isolated by a method such as filtration.

(2) separation of the solid mixture of cultured cells and yellow crystal precipitate from the liquid medium, for example by centrifuging the culture mixture, or by filtering the culture mixture using a nylon or metal filter with appropriate pore size; death,
The solid mixture is treated with a solvent such as chloroform, methanol, ethanol, etc., and berberine is extracted into the solvent, and berberine is isolated by crystallization. On the other hand, berberine is dissolved in the previously separated liquid, but this is for example Am
After adsorption on an ion exchange resin such as berlite XAr1-2, berherin 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.

[Effects of the Invention] If tissue culture is performed using cultured Japanese larch cells having the ability to release berberine obtained by the method of the present invention, more berberine can be produced than in the conventional method, and the cultured cells cannot be disrupted as in the conventional method. Since no treatment is required, the process of separating berberine is simplified and is industrially useful.

〔Example〕

The content of the present invention will be explained in more detail below using examples.

Example 1 Leaves, petioles, and flower buds of Japanese larch were sterilized in a conventional manner,
Calli were induced from these by a conventional method using a Linsmeyer-Skoog solid medium containing 100 μM NAA, 5 μM BA, and 30 g/ff of sucrose.

Since the ability of callus to proliferate and produce berberine differs depending on the tissue of the Japanese larch used, 1. The callus was grown and divided in the dark at 25°C using the above-mentioned Linsmeyer-Skoog solid medium, and the yellow color By selecting dense, well-growing calli and subculturing them for about one year, stable calli with a deep yellow color and a high production of berherin were obtained.

Next, the selected callus was treated with 100 μM NAA and 5 μM BA.
and sucrose at a concentration of 30 g # into a Linsmeyer-Skoog liquid medium at a temperature of 25 °C.
The cells were subcultured under conditions of 1 shaking stroke/min, and cultured cells with deep yellow color and good growth were selected using a Komagome pipette with a thick tip, transferred to a fresh liquid medium, and cultured again.

In this case, the number of culture days after transferring the selected cells to a fresh liquid medium is 2 to 3 weeks, and this selection and subplanting are carried out for 2 to 3 weeks.
By repeating the fermentation process approximately 5 times over a period of 3 months, it was possible to obtain berberine-releasing cultured cells that produced a large amount of berberine and released more than 80% of the produced berberine to the outside of the cells.

Next, the berberine-releasing cultured cells were treated with NAAloo using a 1003 2 1 Erlenmeyer flask.
μM, BA5μM and sucrose at 30g/1
24.2 mg of berberine nitrate could be produced per 30 ml of the medium by culturing for 18 days in a Linsmeyer-Skoog liquid medium containing 25°C and shaking at 100 strokes/min. . Note that 12.4% of the produced berberine was present within the cells, and the rest was present in the medium.

In the medium, some berberine was precipitated as stellate and columnar herherin nitrate.

Comparative Example 1 Rinsmeyer without adding B'A in Example 1
When subculture is carried out in the same manner as in Example 1 except that Skoog's liquid medium is used, pale yellow to white berberine is not produced, or even if it is produced, it contains only a very small amount of berberine. Only cultured cells without berberine-releasing ability were obtained.

4

Claims (1)

[Claims] (1) Using a solid or liquid medium containing benzyladenine, select deep yellow cultured cells among the cultured Japanese larch cells and subculture the cultured cells to release berberine to the outside of the cells. Method for obtaining berberine-releasing cultured cells.