JPS626676A - Method of tissue culture for plant of genus coptis - Google Patents

Abstract

PURPOSE:In producing isoquinoline alkaloid useful as a stomachic, etc., by subjecting a plant of the genus Goptis to tissue culture by using a liquid medium, to improve yield, productivity, etc., by adjusting dissolved oxygen concentration of the medium to a specific range. CONSTITUTION:A plant such as C, japonika Makino var. dussecta Naki, C. japonika Makino var. japonica, etc., belonging to the genus Coptis is subjected to tissue culture by using a liquid medium comprising an inorganic component (e.g., nitrogen, phosphorus or potassium) and a carbon source (e.g., sucrose or ethanol) as essential components blended with a plant hormone, a vitamin and an amino acid. In the operation, dissolved oxygen concentration of the medium is adjusted to 10-20ppm to carry out the tissue culture. Consequently, a large amount of isoquinoline alkaloid such as berberine, etc., can be produced, the culture cell is separated from the liquid medium after the culture is over and extracted with a proper solvent to give advantageously the isoquinoline alkaloid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing isoquinoline alkaloids such as berberine, which are used in stomachic medicines, dyes, etc., by tissue culturing plants of the genus Aureus.

[Conventional technology]

A conventional method for obtaining isoquinoline alkaloids is to directly collect the alkaloids by extraction or the like from naturally grown plants of the genus Orensis. It is not an advantageous method because it takes time and effort to collect plants.

Therefore, as an alternative method, for example, phytochemistry 23@
As described in pages 281 to 285 (1984), a method for obtaining isoquinoline alkaloids such as berberine, palmatine, coptesin, and yateorisine by tissue culture of plants of the genus Aureus has been proposed.

However, these conventional tissue culture methods have a problem in that the yield of the target isoquinoline alkaloid produced from cultured cells is low.

[Problem that the invention seeks to solve]

Therefore, when aiming at industrial production of isoquinoline alkaloids using such tissue culture methods, it has been an important issue to further increase productivity.

In view of these circumstances, the present inventors investigated a method for efficiently culturing the tissues of plants of the genus Orensis to obtain more isoquinoline alkaloids than conventional methods.

[Means for solving problems]

As a result, the present inventors discovered that the productivity of isoquinoline alkaloids in tissue culture is greatly influenced by the dissolved oxygen concentration of the culture medium in tissue culture of plants of the genus Orientalus, which led to the completion of the present invention. Ta. That is, according to the method of the present invention, isoquinoline alkaloids are produced by culturing the tissue culture of a plant of the genus Auris using a liquid medium with a dissolved oxygen concentration of 10 to 20 ppm. tissue culture methods,
is provided.

Examples of plants of the genus Coptis used in the method of the present invention include Coptis japonica.
Makino), C. japoni
Ka Makin.

var, dussecta Nakai), C. var.

japonika Makino var, japon
ica ), C. japonika
Makino var, major 5atake
), Baikaouren CC, quinquefolia
Miq, ) and C. trifol
ia 5alisb, ), etc. In the present invention, among these plants, it is particularly preferable to use Seriba orensis.

The liquid medium used in the present invention is a medium containing inorganic components and a carbon source as essential components, to which are added plant hormones and vitamins, and further added with amino acids as necessary.

Inorganic components of the medium include nitrogen, phosphorus, potassium, sodium, thorium, calcium, magnesium, sulfur, iron,
Manganese, zinc, boron, molybdenum, chlorine, iodine,
Examples include inorganic salts containing elements such as cobalt, specifically potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium l-hydrogen phosphate, sodium 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 medium include carbohydrates such as sucrose and their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of plant hormones in the medium include naphthaleneacetic acid (NAA), indoleacetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and indolebutyric acid (IBM). Examples include auxins such as and derivatives thereof, and cytokinins such as benzyladenine (BA), kinetin, and zeatin. In the present invention, it is usually desirable not to add cytokinins to the culture medium, but when added as necessary, the concentration of cytokinins is usually 10 M.
It is preferable to use it at a low concentration of (0.02 ■/l) or less.

The vitamins in the medium include biotin, thiamine (vitamin B), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and ribobravin (vitamin B2).

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

The medium of the present invention usually contains about 0.1 of the inorganic components.
μM to about 100 mM, about 1 g/z to about 100 g/l of the carbon source, and about 0 g/z of the plant hormones.
．． 1rng/l to about 100mg/l, and about 0.1mg/j of the above vitamins! or about 150mg/I
t and the above amino acids at 0 to about 1000 mg/
It is desirable to use it with 1.

Specifically, the medium used for the tissue culture of plants of the genus Aureus of the present invention may be a conventionally known culture medium used for tissue culture of plants, such as Murashige & Skoo ('62).
g] culture medium, Linsmeyer-Skoog (RM-19
65) (Lins+++aier & Skoog
) medium, white ("63) (White)
, Gamborg's B-5 medium, Mitsui's M-9 medium, Hetch Niche's medium CN1ts
An example is a culture medium prepared by adding the above-mentioned carbon sources and plant hormones to the culture medium (ch & N1tsch), and further adding the above-mentioned vitamins and amino acids as necessary. , Linsmeyer-Skoog, or Murashige-Skoog's medium is preferred. In addition, regarding the composition of the above-mentioned conventionally known culture medium, for example,
It is described in "New Plant Tissue Culture JP P386-P391," written by Furuya, Shokusho Shoten, 1979.

In the present invention, isoquinoline alkaloids are produced by culturing tissues of plants of the genus Aureus using the liquid medium described above. In this case, in the present invention, the dissolved oxygen concentration of the medium is usually 10 to 20 ppm, preferably 10 to 15
Tissue culture is performed in such a way that ppm. When dissolved oxygen concentration is usually lower than 10ppm and usually 20p
If it is higher than pm, it is not preferable because the production amount of inquinoline alkaloids by cultured cells of the genus Aureus decreases. Examples of methods for bringing the dissolved oxygen concentration of the culture medium within the above-mentioned concentration range according to the present invention include the following methods.

In other words, in a liquid medium in which tissue culture is normally carried out at a temperature of 1/2 to 1/2 C, the dissolved oxygen concentration of the medium can be brought into the above range by bringing the medium into contact with oxygen-containing moths that come into contact with the medium by an appropriate method. Specific examples of the oxygen-containing gas in this case include gases containing inert components such as nitrogen and having various oxygen concentrations, and pure oxygen gas.As the oxygen-containing gas, air is used as a culture medium at below atmospheric pressure. The contacting method is the method used in conventional tissue culture of Zboisia plants, and in this method, the dissolved oxygen concentration in the medium varies somewhat depending on the culture temperature, but is usually 8 pp.
Since it is as low as about m, the production amount of isoquinoline alkaloids is small, which is not preferable. In the present invention, air can be used as the oxygen-containing gas if necessary, and in this case, the total pressure in the system is increased by pressurizing the gas to bring the oxygen partial pressure in the gas into the above range. Accordingly, the dissolved oxygen concentration of the medium can be kept within the range of the present invention.

When performing tissue culture under atmospheric pressure, the dissolved oxygen concentration in the medium usually increases by only about 40 ppm even if pure oxygen gas is used, so if you want to further increase the oxygen concentration, use any commonly known appropriate method. A method of culturing tissue in a pressurized system is adopted. In short, in the present invention, culture is carried out with the dissolved oxygen concentration in the culture medium within the above range by appropriately selecting the oxygen partial pressure in the oxygen-containing gas depending on the situation.

In the present invention, it is not necessary to use any particular method for bringing the oxygen-containing gas into contact with the culture medium, and any commonly known method can be employed. Specifically, the contact method includes, for example, an aerated culture method in which oxygen-containing gas is released into the medium through a gas disperser with many holes placed in the liquid medium, or a special method such as silicone, Teflon, etc. Examples include a method in which a gas inlet of an arbitrary shape made of an oxygen-permeable membrane made of a material is brought into contact with the culture medium, and oxygen is supplied into the culture medium through this membrane. In the method using an oxygen permeable membrane,
This method is more preferable because there is no generation of intense air bubbles as is the case with aerated culture, so it is possible to avoid applying undesirable external forces to the culture.

Examples of methods for measuring the dissolved oxygen concentration of a culture medium include the following methods. −−■1−−−Measure the current generated in proportion to the dissolved oxygen concentration using a galpanic cell type oxygen electrode, and automatically reduce the amount of oxygen supplied when the current exceeds a predetermined dissolved oxygen concentration. If the current value decreases and is less than the current value,
This method maintains the dissolved oxygen concentration at a predetermined value by automatically increasing the supply amount.

In the tissue culture carried out in accordance with the present invention, there is no particular need to use a culture tank or a culture device (any device may be used as long as it satisfies the requirements of the present invention described above). In the present invention, the culture medium may be agitated by means such as shaking, swirling, or stirring blades of the culture tank itself, as necessary, or may be allowed to stand still.Alternatively, the present applicant It is also possible to adopt the method of liquid spraying proposed in Japanese Patent Application No. 59-262099, that is, the method of culturing by spraying a liquid medium from above the culture in a shower-like manner.In this case, the liquid medium is An example of a method is to bring the culture medium into contact with an oxygen-containing gas at a location other than the culture vessel to adjust the dissolved oxygen concentration of the culture medium to the above range, and then introduce this liquid culture medium into the culture vessel and reuse it.

In the present invention, isoquinoline alkaloids are produced by tissue culturing plants of the genus Aureus by the method described above. Specific examples of the isoquinoline alkaloid in this case include berberine, palmatine, coptesin, and yateorisine, among which berberine is preferred.

The specific method of tissue culture of the present invention will be described below.

First, the plant body of a plant belonging to the genus Orensis, such as roots,
Tissue pieces collected from growing points, leaves, stems, fruits, seeds, etc., are subjected to, for example, New Plant Tissue Culture (Breakfast Shoten 1979 Edition), 2
Place the plate on Rinsmeyer Skoog's medium (RM-1965) hardened with agar described on page 1,
A part of the tissue piece is formed into a callus by culturing at 35° C. for about 7 to 30 days.

When the callus of a plant of the genus Orenia thus obtained is subcultured by a commonly known method, the growth rate of the callus gradually increases. Next, this callus is grown in a liquid medium suitable for propagation, such as New Plant Tissue Culture (Rikin Shoten 1979).
If the cells are transferred to the Linsmeyer-Skoog liquid medium described in 2010, p. 21 and further grown, the growth rate of the cultured cells will be further increased and stabilized cultured cells will be obtained. In the method of the present invention, the thus obtained stabilized cultured cells are added to the liquid medium of the present invention and further cultured.

The culture temperature in the tissue culture of the present invention is usually as follows:
About 10 to about 35°C, especially about 23 to about 28°C is preferred; when the temperature is less than about 10°C, the growth rate of cultured cells is low, and the same is true when the temperature is raised to 35°C or higher. The growth rate of cultured cells decreases. Although light is not necessarily required for the tissue culture of the present invention, irradiation with light does not inhibit the production of isoquinoline alkaloids such as berberine.

In the method of the present invention, after the culture is completed, the cultured cells are separated from the liquid medium by a method such as decantation or filtration, and then the target isoquinoline alkaloid such as berberine is extracted from the cultured cells using conventionally known methods. It can be separated by methods such as extraction, which are commonly used for natural products such as Oriental apricot. The purity of the alkaloid thus obtained can be increased by a method such as recrystallization, if necessary.

〔Effect of the invention〕

According to the tissue culture method for plants belonging to the genus Aureus of the present invention, more isoquinoline alkaloids such as berberine can be produced than in conventional methods.

〔Example〕

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

Example 1 Coptis japonica M
akin.

Var, dissecta Nakat) leaves 70
% ethanol solution and sodium hypochlorite aqueous solution? After sterilization with & (0.5% available chlorine), a sterile Linsmeyer-Skoog liquid medium containing 3% sucrose, 10 M naphthalene acetate, and 10 M benzyladenine (
Solid medium (composition shown in Table 1) solidified with agar (Agar 1
% by weight) and cultured at 25° C. in the dark to obtain a callus of Seriba aurifolia. Next, this callus of Seriba orensis was planted every 14 days in a Linsmeyer-Skoog liquid medium under the same conditions as above, and the growth rate of the cultured cells of Seriba orensis was determined by rotating it in a rotary shaker using a rotating ring # (amplitude 25+u, 1100 rp). Accelerated and stabilized cultured cells of Seriba ouren were obtained.

An aerated agitation culture tank equipped with a dissolved oxygen concentration meter and an oxygen-containing gas ventilation pipe (inner volume 2 was filled with 1.51 liters of the above sterile liquid medium with the Cu2+ concentration changed to 1 μM and 15 g fresh weight of the above callus, and the dissolved oxygen concentration Adjust the amount of oxygen-containing gas aeration so that
Cultured for a week. The weight of the obtained callus after drying is 1
It was 7.9 g (11.9 g/β). After crushing the dried callus using a mortar, isoquinoline alkaloids were extracted with 90% methanol. When the product extract was analyzed for isoquinoline alkaloids such as berberine, palmatine, coptesin, and yateorigin using high performance liquid chromatography, the total amount of the isoquinoline alkaloids per dry callus weight was 12.7% by weight. Of these, berberine accounts for 8.1% by weight.
In addition, the method of Yamada et al. (Phyt
ochemistry, 23281 (1984)
), the following conditions were used.

Column: μm bonder pack CIB, 3Qcm X3.
9mm melting medium: Sodium octanesulfonate 5mM
and water containing 1% acetic acid - acetonitrile (13nia) Measurement wavelength: 254 nm Examples 2-5, Comparative Examples 1-2

Claims (1)

[Claims] (1) When tissue culturing plants of the genus Orensis using a liquid medium, the dissolved oxygen concentration of the medium should be 10 to 20 pp.
1. A tissue culture method for a plant of the genus Orensis, characterized in that it produces an isoquinoline alkaloid as m.