JPH0746987B2 - Method for tissue culture of Orchid plant - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an isoquinoline alkaloid such as berberine, which is used as a stomachic drug, a dye, etc., by tissue-culturing a plant of the genus Oren.

[Conventional technology]

As a conventional method for obtaining an isoquinoline alkaloid, there is a method of directly collecting the alkaloid from a naturally grown plant of the genus Orenum by extraction or the like, but the method is dependent on the natural environment and weather such as the growth of the plant. Moreover, it is not an advantageous method because it takes time and labor to collect the plants.

Therefore, as an alternative method, for example, as described in Phytochemistry, Vol. 23, pages 281-285 (1984), etc., berberine, palmatin, copthecin, ateoridin etc. A method for obtaining the isoquinoline-based alkaloids has been proposed. However, these conventionally known tissue culture methods have a problem that the yield of the desired isoquinoline alkaloid produced from the cultured cells is low.

[Problems to be solved by the invention]

Therefore, when aiming at the industrial production of isoquinoline alkaloids by such a tissue culture method, it was an important issue to further increase the productivity.

In view of such circumstances, the present inventors have studied a method of efficiently culturing the tissue of a plant of the genus Coptis to obtain more isoquinoline alkaloids than the conventional method.

[Means for solving problems]

As a result, the present inventors have found that, in tissue culture of a plant of the genus Orchid, the productivity of isoquinoline alkaloids by the tissue culture is greatly affected by the dissolved oxygen concentration of the medium, and have completed the present invention. . That is, according to the method of the present invention, in tissue culture of a plant of the genus Coptis, a dissolved oxygen concentration of the medium is adjusted to 10 to 10.
Provided is a tissue culture method for a plant of the genus Orensis, which comprises producing an isoquinoline alkaloid at 20 ppm.

Examples of the Orchidaceae plant used in the method of the present invention include, for example, Coptis japonica Makino, and Ceriobauren (C. japonika Makino var.dussecta Naka).
i), C. japonika Makino var.japonic
a), C. japonica Makino var.major
Satake), baioca lauren (C. quinquefolia Miq.) And honeywort (C. trifolia Salisb.). In the present invention, it is particularly preferable to use Ceriobaren among these plants.

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

Examples of the inorganic component of the medium include inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine and cobalt, 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, manganese sulfate, copper sulfate,
Examples thereof include compounds such as sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

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

Examples of the plant hormones in the medium include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-
D), auxins such as indolebutyric acid (IBA) and their derivatives, and cytokinins such as benzyladenine (BA), kinetin and zeatin. In the present invention, it is preferable that cytokinins are not usually added to the medium, but when added as necessary, cytokinins should be used at a low concentration of usually 10 ⁻⁷ M (0.02 mg / l) or less. Is preferred.

The vitamins in the medium include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinic acid amide and ribobrabin ( vitamin
B ₂ ) etc. can be illustrated.

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 μm of the inorganic component.
M to about 100 mM, the carbon source is about 1 g / l to about 100 g /
l, the plant hormones about 0.1 mg / l to about 100 mg / l,
It is preferable to use the vitamins in an amount of about 0.1 mg / l to about 150 mg / l and the amino acids in an amount of 0 to about 1000 mg / l.

Specific examples of the medium used for tissue culture of the Orchid plant of the present invention include media conventionally used for tissue culture of plants, for example, Murashige Skoog ('62) [Murashige & Skoog ] Medium, Rinsmeier Skoog (RM-1965) [Linsmaier & Skoog] medium, white ('63) [White] medium, Gamborg B-5 medium, Mitsui M-9 medium, Nichichi A medium prepared by adding the above-mentioned carbon source and plant hormones to a Nitsch medium [Nitsch & Nitsch], etc., and further adding the above-mentioned vitamins and amino acids as necessary can be exemplified, but in the present invention, Of these, a medium prepared using a Nichi-Nitchi, Rinsmeier-Skoog or Murashige-Skoog medium is particularly preferable. The composition of the conventionally known medium described above is described, for example, in "New Plant Tissue Culture" P386 to P391 by Takeuchi, Nakajima and Furuya, Asakura Shoten, 1979.

In the present invention, tissue culture of the Orchid plant is performed using the liquid medium described above, and isoquinoline alkaloids are produced, but in this case, the dissolved oxygen concentration of the medium in the present invention is usually 10 to 20 ppm, preferably 10 to 15 ppm. The tissue culture is carried out as follows. Dissolved oxygen concentration is usually 10 ppm
If it is lower than the above range and usually higher than 20 ppm, the amount of isoquinoline alkaloids produced by cultured cells of the genus Coptis decreases, which is not preferable. Examples of the method for adjusting the dissolved oxygen concentration of the medium to the concentration range carried out in the present invention include the following methods. Ie usually 10 or
In a liquid medium in which tissue culture is performed at 35 ° C., the oxygen partial pressure of the oxygen-containing gas contacting the medium is usually 180 mmHg or more,
By contacting the oxygen-containing gas and the culture medium by an appropriate method preferably to 200 mmHg to 800 mmHg,
The dissolved oxygen concentration of the medium can be within the above range. Specific examples of the oxygen-containing gas in this case include gases having various oxygen concentrations containing an inert component such as titanium and pure oxygen gas. A method of contacting air as an oxygen-containing gas with a medium at a body pressure or below is a method adopted in the tissue culture of a conventional Zboisia plant, in which the dissolved oxygen concentration of the medium is slightly different depending on the culture temperature. Is usually as low as about 8 ppm, and the amount of isoquinoline-based alkaloids produced is small, which is not preferable. In the present invention, air can be used as the oxygen-containing gas as necessary, and in this case, the total pressure in the system is increased by pressurizing the gas so that the oxygen partial pressure in the gas falls within the above range. By doing so, the dissolved oxygen concentration of the medium can be within the range of the present invention. When performing tissue culture under atmospheric pressure, the dissolved oxygen concentration of the medium usually increases only about 40 ppm even if pure oxygen gas is used, so if you want to further increase the oxygen concentration, use a generally known appropriate method. Therefore, a method of performing tissue culture in a pressurized system is adopted. In short, according to the present invention, the dissolved oxygen concentration of the medium is adjusted to the above range to carry out the culture 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 method for bringing the oxygen-containing gas into contact with the culture medium, and any conventionally known method can be adopted. As the contact method, specifically, for example, an aeration culture method in which an oxygen-containing gas is released into the medium through a gas disperser having a large number of holes placed in the liquid medium, or a special method such as silicon or Teflon. It is possible to exemplify a method in which a gas inlet having an arbitrary shape made of an oxygen permeable membrane made of a material is brought into contact with a medium and oxygen is supplied into the medium through the membrane. In the method using the oxygen permeable membrane,
It is more preferable because it is possible to prevent undesired external force from being applied to the culture, since vigorous bubbles are not generated unlike in aeration culture.

As a method for measuring the dissolved oxygen concentration of the medium, there are the following methods, for example. The galvanic cell type oxygen electrode is used to measure the current generated in proportion to the dissolved oxygen concentration, and when the current exceeds the current corresponding to the predetermined dissolved oxygen concentration, the supplied oxygen amount is automatically reduced, and below the current value. In this case, the dissolved oxygen concentration of the medium is maintained at a predetermined value by automatically increasing the supply amount.

Regarding the tissue culture carried out in the present invention, it is not necessary to use any particular culture tank or culture apparatus, and any one can satisfy the requirements of the present invention described above. But you can use it.
In the present invention, the medium, if necessary, shaking of the culture tank itself,
It may be stirred by means such as swirling or stirring blades, or may be left stationary. Alternatively, the applicant filed Japanese Patent Application Sho 59
It is also possible to employ the liquid spraying method proposed by No. 262099, that is, a method of culturing by spraying a liquid medium from above the culture in a shower form. In this case, the liquid medium is brought into contact with an oxygen-containing gas at a place different from that of the incubator to adjust the dissolved oxygen concentration of the medium to the above range, and then the liquid medium is introduced into the incubator for recycling. It can be illustrated.

In the present invention, an Orchid plant is tissue-cultured by the above method to produce an isoquinoline alkaloid. Specific examples of the isoquinoline-based alkaloid in this case include berberine, palmatine, copthecin, yateolidine, and among them, berberine is preferable.

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

First, a plant body of a plant belonging to the genus Coptis, for example, a root,
Tissue pieces collected from growing points, leaves, stems, fruits, seeds, etc., for example, new plant tissue culture (Asakura Shoten 1979 edition), hardened with agar described on page 21, Rinsmeier Skoog medium (RM-1965) placed on the floor, 7 ~ 30 at 10 ~ 35 ℃
By culturing for about one day, a part of the tissue piece is turned into callus. When the callus of the plant of the genus Orchid obtained in this manner is subcultured by a commonly known method, the growth rate of the callus gradually increases. Then, this callus is transferred to a liquid medium suitable for growth, for example, new plant tissue culture (Asakura Shoten, 1979 edition), Rinsmeier Skoog liquid medium described on page 21, and further grown to grow the cultured cells. The speed is further increased and stable cultured cells are obtained. In the method of the present invention, the stabilized cultured cells thus obtained are added to the liquid medium of the present invention and further cultured.

As the culture temperature in the tissue culture of the present invention, usually,
About 10 to about 35 ° C., especially about 23 to about 28 ° C. are particularly preferable, and if the temperature is lower than about 10 ° C., the growth rate of the cultured cells is low, and the same is true when the temperature is 35 ° C. or higher. In addition, the growth rate of cultured cells decreases. In performing the tissue culture of the present invention, light is not always necessary,
Irradiation with light does not prevent the formation 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 desired isoquinoline alkaloids such as berberine are conventionally known. It can be separated by a known method such as extraction applied to natural products such as laurel and rye. The alkaloid thus obtained can be increased in purity by a method such as recrystallization, if necessary.

〔The invention's effect〕

According to the method for tissue culture of a plant of the genus Orchid of the present invention, more isoquinoline alkaloids such as berberine can be produced as compared with the conventional method.

〔Example〕

Hereinafter, the method of the present invention will be described more specifically with reference to Examples.

Example 1 Ceribis japonica Makino Var.dissect
a Nakai) leaves were sterilized with 70% ethanol solution and sodium hypozinc acid aqueous solution (effective chlorine amount 0.5%), and then sucrose 3%, naphthalene acetic acid 10 ^-5 M, benzyladenine 10
^-8 M aseptic Rinsmeier-Skoog liquid medium (composition is shown in Table 1) was placed on agar-solidified solid medium (agar 1% by weight) and cultured at 25 ° C in the dark. Got calli of Ceriba lauren. Next, this callus of Seriobauren was planted every 14 days in a liquid medium of Rinsmeier-Skoog under the same conditions as above, and subjected to swirling culture (amplitude 25 mm, 100 rpm) on a rotary shaker to increase the growth rate of cultured cells of Seriobauren. Accelerated and stabilized cerevisiae cultured cells were obtained.

Dissolved oxygen concentration was added to an aerated stirred culture tank (internal volume 2 l) equipped with a dissolved oxygen concentration and oxygen-containing gas aeration tube (1.5 l of sterile liquid medium with Cu ²⁺ concentration changed to 1 μM and 15 g fresh weight of callus). The culture was carried out at 25 ° C in the dark for 2 weeks while controlling the aeration amount of the oxygen-containing gas so that the concentration was 10 ppm. The weight of the obtained callus after drying was 17.9 g (11.9 g / l). After crushing the dry callus using a mortar, isoquinoline alkaloids were extracted with 90% methanol. When this extract was analyzed for isoquinoline alkaloids such as berberine, palmatine, copthecin, yateolidine using high performance liquid chromatography, the total amount of the isoquinoline alkaloids per dry callus weight was 1
It was 2.7% by weight. Of this, berberine was 8.1% by weight.

In addition, the method of Yamada et al. [Phytoche
mistry, 23 281 (1984)], the following conditions were used.

Column: μ-bonder pack C ₁₈ 30 cm × 3.9 mm Solvent: Water containing 5 mM sodium octanesulfonate and 1% acetic acid-acetonitrile (13: 7) Measurement wavelength: 254 nm Examples 2-5, Comparative Examples 1-2 Dissolved oxygen concentration Table 2 shows the results obtained in the same manner as in Example 1 except that was changed as shown in Table 2.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 5/04 C12R 1:91) C12R 1:91)

Claims (1)

[Claims] 1. When tissue culture of a plant of the genus Coptis is carried out using a liquid medium, the dissolved oxygen concentration of the medium is adjusted to 10 to 20 pp.
A method for tissue culture of a plant of the genus Orenus, which comprises producing an isoquinoline alkaloid as m.